牙鲆HRI多克隆抗体的制备及病毒诱导的组织表达分析  

PREPARATION OF POLYCLONAL ANTIBODY AND VIRUS-INDUCED TISSUE EXPRESSION OF PARALICHTHYS OLIVACEU HRI

作　　者：朱蓉[1] 张义兵[1] 张奇亚[1] 桂建芳[1] 

机构地区：[1]中国科学院水生生物研究所淡水生态与生物技术国家重点实验室中国科学院研究生院,武汉430072

出　　处：《水生生物学报》2007年第1期119-124,共6页Acta Hydrobiologica Sinica

基　　金：973计划项目(2004CB117403);863计划项目(2002AA626010);国家自然科学基金项目(30200207;30471333)资助

摘　　要：血红素调节的eIF2α激酶HRI是一种丝氨酸/苏氨酸蛋白激酶,在血红素缺乏和热休克等胁迫存在时,通过磷酸化底物eIF2α调控真核细胞的蛋白合成。牙鲆PoHRI是在鱼类鉴定的第一个HRI基因。PCR克隆编码PoHRI蛋白1-200氨基酸的cDNA片段,定向插入pET32a构建表达载体。IPTG诱导,随后利用Ni2+-NTA亲和层析柱纯化,成功获得预期大小的融合蛋白。将纯化的融合蛋白免疫小鼠获得抗PoHRI多克隆抗体,抗体中和实验进一步证实了抗PoHRI多克隆抗体对PoHRI蛋白的特异性识别。在健康的牙鲆组织中能检测到PoHRI mRNA和蛋白的组成型表达。人工感染大鳞鲆弹状病毒SMRV诱导相应组织PoHRI的表达明显上调。结果表明,PoHRI是一种在牙鲆组织中广泛表达的蛋白,可能在抗病毒免疫反应中发挥某种功能。The heme-regulated initiation factor 2a （elF2a） kinase （HRI） is one of Ser/Thr kinases that phosphorylate the a-subunit of elF2 to inhibit protein synthesis in eukaryotic cells under various stress conditions, including heme deficiency and heat shock. HRI is firstly discovered in rabbit reticulocytes as an inhibitor of globin synthesis under heme deficiency, but so far little is known about the importance of HRI in stress conditions other than heme deficiency and the roles of HRI in nonerythroid tissues. For example, there was an argument on the tissue specificity of HRI expression due to the fact that no HRI protein expression was reported in nonerythroid tissues and a finding that in HRI knockout mice, only red blood cells （RBCs） and their precursors were directly affected by the lack of HRI. Paralichthys olivaceu HRI is the first identified fish homologue, and a previous study showed for the first time that Po HRI could be induced by virus infection in vitro. In order to further investigate whether PoHRI protein is also synthesized in nonerythroid tissues and can be induced by virus infection in vivo, here a anti-PoHRI polyclonal antibody was prepared and subsequently used to detect the expression of PoHRI in different tissues of flounder. Firstly, the cDNA fragment encoding 1-200 amino acids of PoHRI was cloned and integrated into a prokaryotic expression vector pET32a. Next, a recombinant protein similar to the expected size was induced and then purified by Ni2^＋ -NTA chromatography. The anti-Po HRI polyclonal antibody was then prepared by immunization of mice. The specific recognition of anti-PoHRI polyclonal antibody to PoHRI was further verified by a test in which anti-PoHRI antiserum, after pre-adsorbed with recombinant PoHRI peptide, cannot recognize the corresponding polypeptide. In healthy flounder tissues, the constitutive expression of PoHRI was able to be detected at both mRNA and proteins levels, and SMRV infection was able to induce the upregulation of Po HRI in the corresponding ti

关 键 词：HRI 原核表达 多克隆抗体 病毒感染 诱导表达 

分 类 号：S941[农业科学—水产养殖]